An In Vitro Method to Generate Astrocyte Scaffolds within Hydrogel Micro-columns

Take a culture dish containing a micro-column boat comprising hollow tubes of defined diameter.      

The micro-columns, made of a hydrogel, are filled with a buffer to prevent drying.

Under a stereomicroscope, remove the buffer and add an ice-cold collagen solution, a component of the extracellular matrix.   

Introduce a media along the dish's inner edge to maintain column hydration. Incubate under physiological conditions for the collagen to polymerize, forming a coating inside the micro-columns.         

Add astrocytes inside the micro-columns and incubate to promote astrocyte adhesion to collagen.

Next, fill the dish with the medium and maintain it under physiological conditions.       

Due to the micro-columns' narrow diameter, the astrocytes acquire a bipolar morphology with processes extending along the length.

Over time, the astrocytes self-assemble to form a three-dimensional scaffold containing dense bundles of astrocyte processes.

The hydrogel-encased astrocyte scaffold is ready for downstream assays.       

Inside a biosafety cabinet, prepare a 1 milligram per milliliter solution of rat tail type I collagen in co-culture medium and then place it on ice. Adjust the pH of the ECM solution with acid or base as necessary, until the pH is stable in the 7.2 to 7.4 range. Transfer the microcolumn boat to a 35 or 60-milliliter Petri dish with forceps. Using the stereoscope for guidance, situate the 10-microliter tip of a micropipette at one end of each microcolumn and suction to empty the lumen of DPBS and air bubbles.

Charge a P10 micropipette with the collagen solution. Place the 10-microliter tip against one end of each microcolumn, and deliver enough solution to fill the lumen, then pipette a reservoir of ECM on either end of the microcolumn. Once all columns have been filled, pipette co-culture medium in a ring around the Petri dish to prevent the columns from drying out.

Incubate the dish containing the microcolumns at 37 degrees Celsius and 5% carbon dioxide for at least one hour to promote polymerization of collagen before adding cells. Afterwards, place the tip of a P10 micropipette at one end of a microcolumn, and transfer approximately 5 microliters of cell solution into the lumen to fill it. After seeding all the microcolumns in a boat, incubate it at 37 degrees Celsius and 5% carbon dioxide for one hour to promote the attachment of astrocytes to the ECM.

After the incubation period, carefully fill the dishes containing the seeded microcolumns with 3 or 6 milliliters of co-culture media for 35 or 60-millimeter Petri dishes, respectively. Maintain the plated microcolumns in culture at 37 degrees Celsius and 5% carbon dioxide to promote the self-assembly of the aligned astrocytic bundles, which should form a bundled cable-like structure after 6 to 10 hours.